1. Field of the Invention
The present invention relates to a method for measuring holoceruloplasmin concentration. More specifically, the present invention relates to a method for measuring holoceruloplasmin concentration on a blood spot using a standard curve of concentration obtained through an enzyme-linked immunosorbent assay (ELISA) or a dissociation-enhanced time-resolved fluoroimmunoassay using a specific polyclonal antibody and a monoclonal antibody of the holoceruloplasmin.
The method for measuring holoceruloplasmin concentration on a blood spot is very useful for screening of Wilson""s disease in population.
2. Description of the Related Art
It was back in 1912 when Wilson""s disease was known to the public for the first time. Now, one in 25,000 to 30,000 people is suffering from an autosomal recessive disease, one of common genetic diseases with a carrier rate of 1/90. Wilson""s disease is copper metabolism defect, that is, copper is not easily excreted through a biliary nor is incorporated into ceruloplasmin. Therefore, copper gets precipitated in the liver, brain, cornea, red blood cell and kidney, causing hepatic dysfunction including hepatitis, liver cirrhosis, neurologic disturbance like dysarthria, hemolytic anemia or renal tubular dysfunction, etc.
Unfortunately, most patients with Wilson""s disease are diagnosed after liver cirrhoisis or neurologic damage already developed. Although liver transplantation is available, many of them suffer to die or live with severe neurologic sequelae. However, if the diagnosis is made at an early stage of clinical course, they can have normal life back by starting an early treatment with an orally applied chelating agent, e.g., D-phenicillamine or trienthylene tetramine, without any further complications. Therefore, early diagnosis and treatment is very important to Wilson""s disease.
One of the most valuable indexes to diagnose Wilson""s disease is level of cerulaplasmin in blood. Ceruloplasmin has a molecular weight of 132,000 and 6-7 atoms of copper per molecule. Normally, ceruloplasmin is plasma protein and contains 95% of the total circulating copper in a body. It also performs various functions, for example, carrying copper to internal tissues, promoting aromatic amine oxidase activation and antioxidation, eliminating free radicals and hydrogen peroxide (H2O2), and controlling inflammatory reaction. In general, a normal person has 20-50 mg/dl of ceruloplasmin in the serum, but Wilson""s disease patients have reduced level of ceruloplasmin. Normally, ceruloplasmin has an oxidase activity and exists as a copper-containing form. Also, normal persons excrete copper efficiently and have little apoceruloplasmin without the oxidase activity inside of the body (3.3xc2x13.1 mg/dl). In contrast, Wilson""s disease patients, in spite of having the equal amount of apoceruloplasmin to that of the normal persons, have little holoceruloplasmin (2.7xc2x12.0 mg/dl).
Therefore, it is very important to quantify the ceruloplasmin in blood, particularly holoceruloplasmin, to diagnose Wilson""s disease. However, in case of neonates, the screening test is ineffective to trace any sign of the disease since they normally have a low ceruloplasmin concentration. Generally, the best time to conduct the screening test would be at an age of 2 to 5, the time the ceruloplasmin concentration becoming as high as that of adults.
The conventional method for measuring ceruloplasmin in the related art includes a method for measuring holoceruloplasmin through oxidase activity of ceruloplasmin using a serum specimen, a radial immunodiffusion assay using a polyclonal antibody, or an immunoturbidimetric assay.
However, the method for measuring ceruloplasmin by measuring oxidase activity of ceruloplasmin is not very effective since ceruloplasmin does not have its own specific substrate. Also, the radial immunodiffusion assay using a polyclonal antibody is not considered specific because the polyclonal antibody reacts to not only holoceruloplasmin but also apoceruloplasmin,
Besides, the above-described methods require a large amount of blood to conduct the assays, and the sera in the blood need to be separated from the whole blood by centrifugation. Even after the sera are obtained, samples need to be frozen before carrying or storing to prevent the degradation of protein in the sera. Therefore, the methods in the related art are not the best way to perform the mass screening test in that they require extra work for collecting, carrying and storing the specimen. Since the number of the specimen obtained at once is limited, a nationwide screening test requiring a large number of the specimen could not be carried out with the conventional methods.